The goal of this study is to capitalize on a recently identified dopamine (DA) transporter (DAT)-dependent phenotype in the nematode C. elegans, termed Swimming-Induced Paralysis (SWIP), to identify proteins involved in the localization and regulation of cocaine and amphetamine-sensitive DAT proteins at presynaptic nerve terminals. In vertebrates, DA modulates multiple behavioral processes involved with locomotion, cognition, and reward. Reuptake through DAT is the primary mechanism by which DA signaling is terminated and is a critical determinant of presynaptic DA homeostasis. Polymorphisms in human DAT have been associated with ADHD, schizophrenia, and drug addiction, and DAT is a major target of cocaine and amphetamine. Heterologous expression studies indicate that DATs are regulated via accessory proteins and kinase-linked signaling pathways that ultimately control transporter localization and activity. Moreover, signaling networks linked to PKC, CaMKII, PI3K, and Akt have been linked to the presynaptic actions of amphetamine and cocaine, revealing many potential new targets for addiction risk and therapeutic development. The technical challenges presented by the study of DAT regulation in the mammalian CNS have encouraged our laboratory to pursue a characterization of DAT-associated phenotypes in C. elegans, where forward genetic screens for DAT modulators are attainable. Previously, our laboratory showed that the C. elegans DAT (DAT-1) is 43% identical to mammalian DATs, is expressed exclusively in DA neurons and terminals, preferentially transports DA, and is sensitive to both cocaine and amphetamine. Under the support of a postdoctoral NRSA, I identified a phenotype, termed Swimming Induced Paralysis (SWIP) that is a reporter of functional DAT-1 expression. Thus, SWIP in dat-1 knockout or cocaine/amphetamine treated animals is reversed when DA synthesis, release, and post-synaptic signaling through the DA receptor DOP-3 are precluded. Based on these data, I launched a pilot forward genetic screen to identify DA-dependent SWIP mutants and have identified two lines bearing novel dat-1 point mutations that cause biosynthetic, trafficking and functional defects in vitro and in vivo as well as several lines where mutations appear to lie in other genes. Building on this effort, I propose the following Specific Aims: 1) To expand the screen for DA-dependent SWIP phenotypes, validating DAT deficiency via dopamine receptor deficiency complementation tests, responsivity to cocaine/amphetamine and DA transport assays on embryonic cultures and 2) To identify non-DAT, SWIP-generating genes using Illumina-based, high-throughput cDNA sequencing with transcriptome profiling, followed by a bioinformatics based elucidation of human homologs. These studies provide a powerful and unique opportunity for insight into presynaptic mechanisms controlling DAT-dependent DA signaling. PUBLIC HEALTH RELEVANCE: Alterations in dopaminergic (DA) neurotransmission are centrally involved in psychostimulant response and addiction. The presynaptic DA transporter (DAT) is the primary mode by which DA signaling is terminated and is a direct target for cocaine and amphetamine. This research engages a powerful genetic model system to elucidate genes that regulate DA signaling and DAT activity with opportunities to identify novel targets for addiction risk and/or treatment.